The bioassay-guided fractionation of the aril of Myristica fragrans (mace spice) yielded five phenolic compounds, one new acyclic bis phenylpropanoid (1) and four previously known phenolic compounds: compounds (1) (S) 1-(3,4,5-trimethoxyphenyl)-2-(3-methoxy-5-(prop-1-yl) phenyl)-propan-1-ol, (2) benzenemethanol; α-[1-[2,6-dimethoxy-4-(2-propen-1-yl)phenoxy]ethyl]-3,4-dimethoxy-1-acetate, (3) odoratisol A, phenol, 4-[(2S,3S)-2,3-dihydro-7-methoxy-3-methyl-5-(1E)-1-propenyl-2-benzofuranyl]-2,6-dimethoxy, (4) 1,3-benzodioxate-5-methanol,α-[1-[2,6-dimethoxy-4-(2-propenyl)phenoxy]ethyl]-acetate, (5) licarin C; benzofuran,2,3-dihydro-7-methoxy-3-methyl-5-(1E)-1-yl-2-(3,4,5-trimethoxyphenyl). An NMR tube Mosher ester reaction was used in an approach to characterize and determine the assignment of the absolute configuration of the new isolated chiral alcohol (1). The PARP-1 inhibitory activity was evaluated for compound (1) (IC50=3.04μM), compound (2) (IC50=0.001μM), compound (4) (IC50=22.07μM) and compound (5) (IC50=3.11μM). Furthermore, the isolated secondary metabolites were tested for NF-κB and K-Ras inhibitory activities. When tested in the p65 assay, compounds (2) and (4) displayed potent NF-κB inhibition (IC50=1.5 nM and 3.4nM, respectively).

Carbon fiber (CF) is predominantly produced from fossil-based sources and is therefore an area of interest for further development towards a more sustainable society. The purpose of this thesis work was to investigate the possibility of producing precursor fibers (PFs) for CF production from a blend of renewable cellulose andlignin. Cellulose, which is used to some extent for CF production, was chosen, while the possibility of adding lignin was investigated in hope of increasing the gravimetric yield of the CF production. Blends of softwood kraft cellulose pulp (SKP) and softwood kraft lignin (SKL) were dissolved in an alkaline (NaOH) solvent system at different cellulose/lignin ratios. A total of eight dopes were prepared (SKP/SKL ratios of 100/0–60/40 wt./wt.) with total dope concentrations ranging from 4.5 wt.% to 9.2 wt.%. The addition of SKL resulted in dark colored dopes with viscosities of which mainly appeared to depend on the SKP concentration. The dopes were wet spun, resulting in continuously spun PFs. The PFs showed on an increasing pyrolysis yield with increased SKL content but decreasing mechanical properties. However, process optimization was not included in the work, subsequently leading to the assumption that greater values on mechanical properties can be achieved. A pure SKP PF and a SKP-SKL (70/30 wt./wt.) PF were successfully thermally converted into CFs by carbonization at 1000 °C. The PF containing SKL had a total gravimetric yield more than twice as high as the pure SKP PF, 28 wt.% and 12 wt.%, respectively. Thereby, the addition of SKL seems to have a positive impact on the CF yield when utilizing a NaOH(aq) solvent system. This thesis work has become a base for the future work towards the development of CFs from wet spun cellulose-lignin PFs in the NaOH(aq) solvent system.  

The formation of C-C/X bonds is essential for the manufacture of a broad range of chemicals and materials used in areas critical for maintaining quality of life in modern society, e.g. pharmaceuticals, agrochemicals and polymers, and for aspects of research in organic chemistry. The use of catalysts for facilitating these reactions is highly desirable due to the improvements in energy and atom economies that can potentially be achieved.The primary objective of the thesis was to explore novel approaches for catalysis of C-C/X bond-forming reactions, both through C-H activation. In paper I, at unable cobalt catalyzed C-H activation-driven annulation of benzamides with unsymmetrical diynes was developed, where either 3- or 4-substitution of the isoquinolone could be steered by the nature of the diyne used. Anunprecedented iridium catalyzed tandem bis-arylsulfenylation of indoles was described (paper II), where an adamantoyl sacrificial directing group plays a key role in the simultaneous direction of arylsulfenylation to the 2- and 4- positions. In paper III, a flow reactor in a lab-on-a-chip device was developed for the Suzuki cross-coupling reaction. Miniaturization provides the opportunity to reduce material consumption. Polyethyleneimine (PEI)-brushes were used for the immobilization of Pd-nanoparticles, and high efficiencies were observed. Collectively, the research underpinning this thesis provides new strategies forC-C and C-X(S) bond formation.

Salt-bridge interactions between acidic and basic amino acids contribute to the structural stability of proteins and to protein-protein interactions. A conserved salt-bridge is a canonical feature of the alpha-defensin antimicrobial peptide family, but the role of this common structural element has not been fully elucidated. We have investigated mouse Paneth cell alpha-defensin cryptdin-4 (Crp4) and peptide variants with mutations at Arg(7) or Glu(15) residue positions to disrupt the salt-bridge and assess the consequences on Crp4 structure, function, and stability. NMR analyses showed that both (R7G)-Crp4 and (E15G)-Crp4 adopt native-like structures, evidence of fold plasticity that allows peptides to reshuffle side chains and stabilize the structure in the absence of the salt-bridge. In contrast, introduction of a large hydrophobic side chain at position 15, as in (E15L)-Crp4 cannot be accommodated in the context of the Crp4 primary structure. Regardless of which side of the salt-bridge was mutated, salt-bridge variants retained bactericidal peptide activity with differential microbicidal effects against certain bacterial cell targets, confirming that the salt-bridge does not determine bactericidal activity per se. The increased structural flexibility induced by salt-bridge disruption enhanced peptide sensitivity to proteolysis. Although sensitivity to proteolysis by MMP7 was unaffected by most Arg(7) and Glu(15) substitutions, every salt-bridge variant was degraded extensively by trypsin. Moreover, the salt-bridge facilitates adoption of the characteristic alpha-defensin fold as shown by the impaired in vitro refolding of (E15D)-proCrp4, the most conservative salt-bridge disrupting replacement. In Crp4, therefore, the canonical alpha-defensin salt-bridge facilitates adoption of the characteristic alpha-defensin fold, which decreases structural flexibility and confers resistance to degradation by proteinases.

The inducible Mar phenotype of Escherichia coli is associated with increased tolerance to multiple hydrophobic antibiotics as well as some highly hydrophobic organic solvents such as cyclohexane, mediated mainly through the AcrAB/TolC efflux system. The influence of water miscible alcohols ethanol and 1-propanol on a Mar constitutive mutant and a mar deletion mutant of E. coli K-12, as well as the corresponding strains carrying the additional acrAB deletion, was investigated. In contrast to hydrophobic solvents, all strains were killed in exponential phase by 1-propanol and ethanol at rates comparable to the parent strain. Thus, the Mar phenotype does not protect E. coli from killing by these more polar solvents. Surprisingly, AcrAB does not contribute to an increased alcohol tolerance. In addition, sodium salicylate, at concentrations known to induce the mar operon, was unable to increase 1-propanol or ethanol tolerance. Rather, the toxicity of both solvents was increased in the presence of sodium salicylate. Collectively, the results imply that the resilience of E. coli to water miscible alcohols, in contrast to more hydrophobic solvents, does not depend upon the AcrAB/TolC efflux system, and suggests a lower limit for substrate molecular size and functionality. Implications for the application of microbiological systems in environments containing high contents of water miscible organic solvents, e. g., phage display screening, are discussed.

N-Iodosuccinimide (NIS) in pure trifluoroacetic acid (TFA) offers a time-efficient and general method for the iodination of a wide range of mono-and disubstituted benzenes at room temperature, as demonstrated in this paper. The starting materials were generally converted into mono-iodinated products in less than 16 hours at room temperature, without byproducts. A few deactivated substrates needed addition of sulfuric acid to increase the reaction rate. Another exception was methoxybenzenes that preferentially were iodinated by NIS in acetonitrile with only catalytic amounts of TFA.

Hydrolysis of labile esters of beta-hydroxyketones has been performed with whole plant tissue from various vegetables. The pheromone 5-hydroxy-4-methyl-3-heptanone (1) was used as the model compound. Hydrolysis of acetates and benzoates of 1 was unsuccessful using normal conditions of ester hydrolysis, both by chemical hydrolysis and by the means of commercial lipases. When, however, whole cells of carrot, celery root, eggplant, parsley root, parsnip and potato were used as reagents, hydrolysis of the acetates was successful. At low conversion the hydrolysis was stereoselective and at total conversion virtually no formation of by-products was observed. The selectivity varied among the eight vegetables that were evaluated. Methods of preparation and substrate-to-plant ratio were examined. Furthermore, acetates and benzoates of three analogous compounds [5-hydroxy-3-heptanone (2), 5-hydroxy-5-methyl-3-heptanone (3) and 5-ethyl-6-hydroxy-4-octanone (4)] were hydrolyzed by potato and sweet potato to various degrees, indicating that the method is general for the mild and stereoselective hydrolysis of secondary beta-alkoxy-and beta-aryloxyketones.

All four possible stereoisomers of 5-hydroxy-4-methyl-3-heptanone were synthesized from common achiral reagents using fast, straightforward organic synthesis, including the use of whole tissue of Daucus carota, Solanum melongena, and Pleurotus ostreatus.

Caeruleanone A (1), a novel rotenoid with an unprecedented arrangement of the D-ring, was isolated with another two new analogues, caeruleanones B (2) and C (3), together with 11 known rotenoids from the fruits of Millettia caerulea. The structures of the new compounds were determined by spectroscopic data analysis, with that of 1 being confirmed by single-crystal X-ray diffraction. Compounds 2 and 3 displayed potent mitochondrial transmembrane potential inhibitory and quinone reductase induction activities.

A small library of nonhydrolyzable mimics of GM1 ganglioside, featuring galactose and sialic acid its pharmacophoric carbohydrate residues,, was synthesized and tested. All compounds were synthesized from readily available precursors using high-performance reactions, including click chemistry protocols, and avoiding O-glycosidic bonds. Sonic of the most active molecules also feature a point of further derivatization that can be used for conjugation will, polyvalent aglycons. Their affinity towards cholera toxin was assessed by weak affinity chromatography, which allowed a systematic evaluation and selection of the best candidates. Affinity could be enhanced up to one or two orders of magnitude over the affinity of the individual pharmacophoric sugar residues.

A cobalt-catalysed, ortho-directed, C-H activation of 8-aminoquinoline amides for the preparation of functionalised isoquinolones is reported. The C-H activation was performed with the amide derived from 8-aminoquinoline which acts as a bidentate directing group to facilitate the C-H activation at the ortho carbon atom of the amide towards annulation/cyclisation, with unsymmetrical 1,3-diynes. The work presented here is an exploration of the regiochemical outcome of an efficient and a novel route of synthesis that tries to gain a deeper insight into the regioselective preference for C-H activated annulations that result in the formation of a diverse range of alkynylated regioisomeric heterocycles. Of the four possible regioisomers, only one is formed as the major product depending on the stereoelectronic properties of the diyne in combination with the nature of the 8-aminoquinoline amide.

The regiochemical outcome of a cobalt(ii) catalysed C-H activation reaction of aminoquinoline benzamides with unsymmetrical 1,3-diynes under relatively mild reaction conditions can be steered through the choice of diyne. The choice of diyne provides access to either 3- or 4-hydroxyalkyl isoquinolinones, paving the way for the synthesis of more highly elaborate isoquinolines.

Background: The interaction between biotin and avidin is utilized in a wide range of assay and diagnostic systems. A robust material capable of binding biotin should offer scope in the development of reusable assay materials and biosensor recognition elements. Results: Biotin-selective thin (3-5 nm) films have been fabricated on hexadecanethiol self assembled monolayer (SAM) coated Au/quartz resonators. The films were prepared based upon a molecular imprinting strategy where N, N'-methylenebisacrylamide and 2-acrylamido-2-methylpropanesulfonic acid were copolymerized and grafted to the SAM-coated surface in the presence of biotin methyl ester using photoinitiation with physisorbed benzophenone. The biotinyl moiety selectivity of the resonators efficiently differentiated biotinylated peptidic or carbohydrate structures from their native counterparts. Conclusions: Molecularly imprinted ultra thin films can be used for the selective recognition of biotinylated structures in a quartz crystal microbalance sensing platform. These films are stable for periods of at least a month. This strategy should prove of interest for use in other sensing and assay systems.

Three varieties of honey of different dominant floral origin were found to attract social Hymenoptera, including the large earth bumble bee, Bombus terrestris, in a New Zealand mountain beech forest. This study was undertaken to identify volatile organic compounds that induce the attraction of bumble bees to honeybee (Apis mellifera) honey. We analyzed the chemical composition of the volatile organic compounds produced in three distinct varieties of honey (i.e., manuka, honeydew, and clover honey). The composition of the chemical profile of the three honey varieties differed in the quality and in the ratio of compounds in the headspace. o-Methoxyacetophenone was the main compound in the headspace of all three honey varieties. Among the 40 compounds identified in the headspace in the three varieties, only seven shared compounds (i.e., benzaldehyde, benzyl alcohol, phenylacetaldehyde, 2-phenylethanol, isophorone, 4-oxoisophorone, and o-methoxyacetophenone) were present in the headspace of the three honey varieties. The relative attractiveness of various blends of the seven common compounds found in the three honey varieties was tested for the attraction to bumble bees in a mountain beech forest. A binary blend of isophorone and 4-oxoisophorone at a ratio of 90:10 was the most attractive blend for both bumble bee workers and queens. A small number of honey bee workers were also attracted to the former binary blend. Our study represents the first identification of a honey-derived attractant for bumble bees and honey bees. The potential application of our finding for monitoring of bumble bees or to enhance crop pollination and help to tackle the current concern of a global pollination crisis is discussed.

The composition of the rectal gland secretion and volatiles emitted by female Queensland fruit fly, Bactrocera tryoni was investigated. Esters were found to be the main compounds in the gland extracts and headspace, while amides were the minor compounds in the gland extracts and headspace. Ethyl dodecanoate, ethyl tetradecanoate, ethyl (Z9)-hexadecenoate and ethyl palmitate were the main esters in the gland extracts, while ethyl dodecanoate and ethyl tetradecanoate were the main esters in the headspace. Four amides (N-(3-methylbutyl)acetamide), N-(2-methylbutyl)propanamide, N-(3-methylbutyl)propanamide, and N-(3-methylbutyl)-2-methylpropanamide were found in the gland extracts and the headspace. Among the amides, N-(3-methylbutyl)acetamide and N-(3-methylbutyl)propanamide were the main amides in the gland extracts and the headspace.Traces of three spiroacetals were found both in the gland extracts and in the headspace. (E,E)-2,8-Dimethyl-1,7-dioxaspiro[5.5]undecane, (E,E)-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane, (E,E)-2-propyl-8-methyl-1,7-dioxaspiro[5.5]undecane. All compounds found in the headspace were present in the extract of the rectal gland suggesting that the rectal gland is the main source of the headspace volatiles, whose function remains to be elucidated.This is the first comprehensive chemical analysis of the rectal gland secretions and volatiles of female B. tryoni, and further laboratory and field bioassays are required to determine the function of compounds identified in this study. Discovery of the same amides previously identified in the male rectal gland in the female rectal gland raises questions about the pheromonal role previously suggested for these compounds.

BACKGROUND: The common wasp, Vespula vulgaris (L.), and the German wasp, Vespula germanica (F.), are significant problems in New Zealand beech forests (Nothofagus spp.), adversely affecting native birds and invertebrate biodiversity. This work was undertaken to develop synthetic attractants for these species to enable more efficient monitoring and management. RESULTS: Seven known wasp attractants (acetic acid, butyl butyrate, isobutanol, heptyl butyrate, octyl butyrate and 2,4-hexadienyl butyrate) were field tested, and only heptyl butyrate and octyl butyrate attracted significantly higher numbers of wasps than a non-baited trap. Accordingly, a series of straight-chain esters from methyl to decyl butyrate were prepared and field tested for attraction of social wasps. Peak biological activity occurred with hexyl butyrate, heptyl butyrate, octyl butyrate and nonyl butyrate. Polyethylene bags emitting approximately 18.4-22.6 mg day(-1) of heptyl butyrate were more attractive than polyethylene bags emitting approximately 14.7-16.8 mg day(-1) of heptyl butyrate in the field. Electroantennogram (EAG) studies indicated that queens and workers of V. vulgaris had olfactory receptor neurons responding to various aliphatic butyrates. CONCLUSION: These results are the first to be reported on the EAG response and the attraction of social wasps to synthetic chemicals in New Zealand beech forests and will enable monitoring of social wasp activity in beech forests. (C) 2009 Society of Chemical industry

The short-chain chloroparaffins (SCCP), (C10-13 chloroalkanes) are identified in the European Water Framework Directive, as priority hazardous substances. Within the ScorePP project, the aim is to develop emission control strategies that can be employed to reduce emissions from urban areas into receiving waters. Six different scenarios for mitigating SCCP emissions in two different semi-hypothetical case cities representing eastern inland and northern coastal conditions have been evaluated. The analysis, associated with scenario uncertainty, indicates that the EU legislation, Best Available Technologies (BAT) and stormwater/CSO management were the most favorable in reducing emissions into the environment.

Rhodnius prolixus is one of the most important vectors of Chagas disease in Central and South America for which repellents and attractants are sorely needed. Repellents like DEET, picaridin, and IR3535 are widely used as the first line of defense against mosquitoes and other vectors, but they are ineffective against R. prolixus. Our initial goal was to identify in R. prolixus genome odorant receptors sensitive to putative sex pheromones. We compared gene expression of 21 ORs in the R. prolixus genome, identified 4 ORs enriched in male (compared with female) antennae. Attempts to de-orphanize these ORs using the Xenopus oocyte recording system showed that none of them responded to putative sex pheromone constituents. One of the them, RproOR80, was sensitive to 4 compounds in our panel of 109 odorants, namely, 2-heptanone, gamma-octalactone, acetophenone, and 4-methylcychohexanol. Interestingly, these compounds, particularly 4-methylcyclohexanol, showed strong repellency activity as indicated not only by a significant decrease in residence time close to a host, but also by a remarkable reduction in blood intake. 4-Methylcyclohexanol-elicited repellency activity was abolished in RNAi-treated insects. In summary, our search for pheromone receptors led to the discovery of repellents for R. prolixus.

Cadmium is a highly toxic group XII metal, similar to zinc and mercury. Unlike zinc, which is one of the most common metal cofactors in biology, cadmium is highly toxic. Many Zn2+-binding proteins can bind Cd2+-ions without significantly affecting their structures. Here, the protein data bank is analysed with regard to protein-cadmium interactions, which shows that cadmium can bind to a variety of ion binding sites in proteins. Statistical analysis of Cd2+-side chain interactions is compared with a similar analysis of other ions. This analysis reveals that with regard to amino acid side-chain preference, Cd2+ is more similar to Mn2+ than to Zn2+ or Hg2+. Finally, the interaction energies of three native metal binding proteins are calculated where Cd2+ binds instead of Zn2+, Ca2+ or Cu2+. The interaction energies are decomposed into individual components whose contributions are discussed.

Herein we report the asymmetric syntheses of a number of oxygenated terpenoids that are of importance in the chemical ecology of bark beetles. These are pinocamphones, isopinocamphones, pinocarvones, and 4-thujanols (= sabinene hydrates). The camphones were synthesized from isopinocampheol, the pinocarvones from beta-pinene, and the thujanols from sabinene. The NMR spectroscopic data, specific rotations, and elution orders of their stereoisomers on a chiral GC-phase (beta-cyclodextrin) are also reported. This enables facile synthesis of pure compounds for biological activity studies and identification of stereoisomers in mixed natural samples.

Molecularly imprinted thin layers were prepared in silica capillaries by using two different surface polymerization strategies, the first using 4,4-azobis(4-cyanovaleric acid) as a surface-coupled radical initiator, and the second, S-carboxypropyl-S'-benzyltrithiocarbonate as a reversible addition-fragmentation chain transfer (RAFT) agent in combination with 2,2-azobisisobutyronitrile as a free radical initiator. The ability to generate imprinted thin layers was tested on two different polymerization systems: (i) a 4-vinylpyridine/ethylene dimethacrylate (4VP-EDMA) in methanol-water solution with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a template; and (ii) methacrylic acid/ethylene dimethacrylate (MAA-EDMA) in a chloroform solution with warfarin as the template molecule. The binding properties of the imprinted capillaries were studied and compared with those of the corresponding non-imprinted polymer coated capillaries by injecting the template molecule and by measuring its migration times relative to a neutral and non-retained marker. The role of running buffer hydrophobicity on recognition was investigated by studying the influence of varying buffer acetonitrile concentration. The 2,4,5-T-imprinted capillary showed molecular recognition based on a reversed phase mechanism, with a decrease of the template recognition in the presence of higher acetonitrile content; whereas warfarin-imprinted capillaries showed a bell-shaped trend upon varying the acetonitrile percentage, illustrating different mechanisms underlying imprinted polymer-ligand recognition. Importantly, the results demonstrated the validity of affinity capillary electrochromatography (CEC) to screen the binding properties of imprinted layers.

Molecular imprinting is a technique used to design polymeric recognition materials with selectivity for a predetermined structure. The molecular imprinting process generates cavities in the polymer matrix that are complementary in size, shape and functionality to the template-structure. The recognition properties of molecularly imprinted polymers (MIPs) are comparable to those of antibodies and enzymes, which make MIPs utilizable in a wide range of application areas including biomimetic assays and biosensors [1]. Previous studies have shown that the prepolymerization step is central for the establishment of high affinity binding sites in MIPs [2]. However, our understanding of the physical mechanisms underlying MIP formation and template recognition is still limited. With the rapid increase of computational power and the development of suitable software molecular dynamics (MD) simulation methods have become a valuable theoretical tool to aid our understanding of the molecular imprinting process, and even in the development of rational design strategies [2]. Recently the first simulation of a complete prepolymerization mixture was presented [3].

Here we present 10 ns MD simulations of a series of all-component prepolymerization mixtures. The simulated systems were assembled with different molar ratios using the local anaesthetic bupivacaine as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinker, 2,2’-azobis-(2-methylpropionitrile) (AIBN) as the initiator and toluene as the solvent. The simulations were performed using the AMBER (v. 10.0 UCSF, San Francisco, CA) suite of programs (4) and the GAFF [6] force field. Molecular trajectories were evaluated with radial distribution functions and hydrogen bond analysis.

References

1. Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O´Mahony, J.; Whitcombe, J., J. Mol. Recognit. (2006), 19, 106-180
2. Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O´Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron. (2009), 25, 543-552
3. Karlsson, B. C. G.; O´Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc. (2009), 131, 13297-13304
4. Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. Comput. Chem. (2009), 26, 1668-1688
5. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. J. Comput. Chem. (2004), 25, 1157-1174

Molecularly imprinted polymers (MIPs) are polymeric receptors with selectivity for a predetermined structure. The molecular imprinting process generates cavities in a synthetic polymer matrix that are complementary in size, shape and functionality to the template. MIPs exhibit recognition properties analogous to their biological counterparts, such as antibodies, and can be utilized in a wide range of application areas [1]. Nonetheless, the physical mechanisms underlying MIP formation and template recognition are still poorly understood. Molecular dynamics (MD) based computer simulations are a valuable theoretical tool which may be used to aid our understanding of the molecular imprinting process, and even for the development of rational design strategies [2]. Recently the first MD simulation of a complete prepolymerization mixture was presented [3].

In the present work, MD simulations of a series of all-component prepolymerization mixtures were performed, using the local anaesthetic bupivacaine as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinker, 2,2’-azobis-(2-methylpropionitrile) (AIBN) as the initiator and toluene as the solvent. The simulated systems differed in the molar fraction of MAA. Systems were evaluated with radial distribution functions and hydrogen bond analyses. By correlating the results with the rebinding behaviour of a series of synthesized MIPs the importance of the stoichiometry between template, functional monomer and crosslinker was highlighted. The analysis of the MD simulations revealed strong competition for hydrogen bonding between the carbonyl oxygen’s of MAA and EGDMA and the amide proton of bupivacaine. Moreover, the hydrogen bonding contact between EGDMA and bupivacaine remained nearly unaffected by the varied molar fraction MAA in the different systems demonstrating the role of the crosslinker being more important as generally accepted.

References

[1]             Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O´Mahony, J.; Whitcombe, J., J. Mol. Recognit., 19, 106-180 (2006)

[2]            Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O´Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron., 25, 543-552 (2009)

[3]            Karlsson, B. C. G.; O´Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc., 131, 13297-13304 (2009)

Through the rapid increase in computational power and the development of suitable software, molecular dynamics (MD) has become a promising tool for use in the development of molecularly imprinted polymers (MIPs).¹ MD is a computational method based on Newtonian mechanics, which enables the simultaneous simulation of thousands of discrete molecules, and can be used to establish the states of the molecular species present in MIP-prepolymerization mixtures. As detailed understanding of the molecular basis for formation of high affinity MIP sites is still lacking and the physical mechanism underlying specific recognition is still a matter of debate, the use of MD as a tool to investigate MIP-prepolymerization mixtures is highly motivated.¹ Recently the first MD simulation of an all-component prepolymerization mixture was presented, which gave a detailed picture of the underlying monomer-template interactions important for the “molecular memory” in MIPs.²

Here, we present results obtained from a series of MD simulations representing all-component MIP/REF prepolymerization mixtures assembled with differences in stoichiometries of functional and crosslinking monomer. In these mixtures, the local anaesthetic drug bupivacaine was used as a template, methacrylic acid as the functional monomer, ethylene dimethacrylate as crosslinking monomer, 2,2’-azobis-(2-methylpropionitrile) as the initiator and toluene as the solvent. Bupivacaine complexation in each system was evaluated with radial distribution functions and hydrogen bond analyses. By correlating the results with the rebinding behaviour of a series of synthesized bupivacaine-MIPs, the relationship between the degree of crosslinking and MIP-performance was highlighted.

[1] Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O´Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron., 25, 543-552 (2009)

[2] Karlsson, B. C. G.; O´Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc., 131, 13297-13304 (2009)

In this report, principal component analysis (PCA) has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP) recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA)-crosslinked polymers had either methacrylic acid (MAA) or methyl methacrylate (MMA) as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD) simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design.

The search for mates and food is mediated by volatile chemicals. Insects sense food odorants and sex pheromones through odorant receptors (ORs) and pheromone receptors (PRs), which are expressed in olfactory sensory neurons. Molecular phylogenetics of ORs, informed by behavioral and functional data, generates sound hypotheses for the identification of semiochemicals driving olfactory behavior. Studying orthologous receptors and their ligands across taxa affords insights into the role of chemical communication in reproductive isolation and phylogenetic divergence. The female sex pheromone of green budworm mothHedya nubiferana(Lepidoptera, Totricidae) is a blend of two unsaturated acetates, only a blend of both elicits male attraction. Females produce in addition codlemone, which is the sex pheromone of another tortricid, codling mothCydia pomonella. Codlemone also attracts green budworm moth males. Concomitantly, green budworm and codling moth males are attracted to the host plant volatile pear ester. A congruent behavioral response to the same pheromone and plant volatile in two tortricid species suggests co-occurrence of dedicated olfactory channels. In codling moth, one PR is tuned to both compounds, the sex pheromone codlemone and the plant volatile pear ester. Our phylogenetic analysis finds that green budworm moth expresses an orthologous PR gene. Shared ancestry, and high levels of amino acid identity and sequence similarity, in codling and green budworm moth PRs offer an explanation for parallel attraction of both species to the same compounds. A conserved olfactory channel for a sex pheromone and a host plant volatile substantiates the alliance of social and habitat signals in insect chemical communication. Field attraction assays confirm that in silico investigations of ORs afford powerful predictions for an efficient identification of behavior-modifying semiochemicals, for an improved understanding of the mechanisms of host plant attraction in insect herbivores and for the further development of sustainable insect control.

New molecularly imprinted polymers (MIPs), which exhibit specific recognition of ethyl carbamate (EC) have been synthesized and studied. In this process, EC was the template molecule and -cyclodextrin derivatives were employed as functional monomers in the molecular imprinting technique (MIT). An EC molecularly imprinted sensor (EC-MIS) was prepared by using MIT surface modification. The EC-MIS was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. EC detection performance, binding parameters and dynamics mechanism were investigated. The result showed that the synthetic route designed was appropriate and that new MIP and EC-MIS were successfully prepared. The EC-MIS exhibited a good molecular recognition of EC. A linear relationship between current and EC concentration was observed using cyclic voltammetry and the detection limit was 5.86 g L-1. The binding constant (K = 4.75 x 10(6) L mol(-1)) between EC and the EC-MIS, as well as, the number of binding sites (n = 1.48) has been determined. The EC-MIS recognition mechanism for the EC is a two-step process. The sensor was applied for the determination of EC in Chinese yellow wines, and the results were in good agreement with the gas chromatography-mass spectrometry (GC-MS) method.

In order to obtain the biological active compound, alpha-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of a-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of alpha-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (H-1 NMR and C-13 NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained alpha-mangostin was 95.6% as determined by HPLC analysis. The binding of native alpha-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that amangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to alpha-mangostin is 6.4832x10(5) L/mol and 1.4652x10(5) L/mol at 298 K and 7.8619x10(5) L/mol and 1.1582x10(5) L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between alpha-mangostin and HSA or TRF were also obtained by virtue of the Forster theory of non-radiation energy transfer. The effect of alpha-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between amangostin and HSA is hydrophobic interactions, while the main interaction between alpha-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.

Cyclotides are a large family of plant peptides that are characterised by a head-to-tail circular backbone and three disulfide bonds that are arranged in a cystine knot. This unique structural feature, which is referred to as a cyclic cystine knot, gives the cyclotides remarkable stability against chemical and biological degradation. In addition to their natural function as insecticides for plant defence, the cyclotides have a range of bioactivities with pharmaceutical relevance, including cytotoxicity against cancer cell lines. A glutamic acid residue, aside from the invariable disulfide array, is the most conserved feature throughout the cyclotide family, and it has recently been shown to be crucial for biological activity. Here we have used solution-state NMR spectroscopy to determine the three-dimensional structures of the potent cytotoxic cyclotide cycloviolacin O2, and an inactive analogue in which this conserved glutamic acid has been methylated. The structures of the peptides show that the glutamic acid has a key structural role in coordinating a set of hydrogen bonds in native cycloviolacin O2; this interaction is disrupted in the methylated analogue. The proposed mechanism of action of cyclotides is membrane disruption and these results suggest that the glutamic acid is linked to cyclotide function by stabilising the structure to allow efficient aggregation in membranes, rather than in a direct interaction with a target receptor.

The relaxin/insulin superfamily is a group of peptide hormones that consists of ten members in human, namely relaxins 1-3, insulin-like peptides (INSL) 3-6, insulin and insulin-like growth factors (IGF) I-II. These peptides have various functions in the body, such as regulating growth, blood glucose levels,  collagen metabolism, germ cell maturation and appetite. Misregulation of these mechanisms is associated with disease and accordingly they are of interest as potential pharmaceutical targets. Structurally the hormones are characterised by two peptide chains, A and B, which are held together by one intra A-chain and two inter chain disulfide bonds. Four different G-protein coupled receptors (GPCR) called relaxin family peptide receptor (RXFP) 1-4 have been found to respond to stimuli by different relaxin peptides. RXFP3 and RXFP4 are classic peptide ligand GPCRs, whereas RXFP1 and RXFP2 are characterised by a large extracellular leucine rich-repeat domain. Relaxin-3, which is the relaxin family ancestor, is the only relaxin peptide known to be able to bind and activate both subtypes of GPCRs, namely RXFP1, RXFP3 and RXFP4.

The aim of this thesis was to analyse the structure-function relationship of the relaxin ligands and receptors, and to use this information to develop selective ligands for the relaxin receptors, which can be used as drug leads or pharmacological tools for investigating the physiological roles of the RXFPs.

The 3D structures of native INSL5 and relaxin-2 were determined by solution NMR spectroscopy. The peptides showed an insulin/relaxin-like overall fold. A relaxin chimera peptide, consisting of the A-chain from INSL5 and the B-chain from relaxin-3, R3/I5, which has been shown to be selective for RXFP3 and RXFP4 over RXFP1, was also subjected to NMR studies. The R3/I5 peptide maintained an insulin/relaxin-like overall fold, and the relaxin-3 B-chain adopted a conformation identical to that in native relaxin-3, confirming that the activity of R3/I5 can be directly related to its primary sequence. Furthermore, a truncation study was undertaken to ascertain the importance of the termini for structure and function. By using the knowledge generated from the structure-function relationship, a single-chain high affinity RXFP3 selective antagonist was developed.

In conclusion, this thesis has contributed to broaden the knowledge of the structure-function relationship of the relaxin ligands and the development of a selective RXFP3 antagonist, which is currently a drug lead for treatment of neurological disorders including stress and obesity.

Relaxin-3 interacts with high potency with three relaxin family peptide receptors (RXFP1, RXFP3, and RXFP4). Therefore, the development of selective agonist and antagonist analogs is important for in vivo studies characterizing the biological significance of the different receptor-ligand systems and for future pharmaceutical applications. Recent reports demonstrated that a peptide selective for RXFP3 and RXFP4 over RXFP1 can be generated by the combination of the relaxin-3 B chain with the A chain from insulin-like peptide 5 (INSL5), creating an R3/I5 chimera. We have used NMR spectroscopy to determine the three-dimensional structure of this peptide to gain structural insights into the consequences of combining chains from two different relaxins. The R3/I5 structure reveals a similar backbone conformation for the relaxin-3 B chain compared to native relaxin-3, and the INSL5 A chain displays a relaxin/insulin-like fold with two parallel helices. The findings indicate that binding and activation of RXFP3 and RXFP4 mainly require the B chain and that the A chain functions as structural support. RXFP1, however, demonstrates a more complex binding mechanism, involving both the A chain and the B chain. The creation of chimeras is a promising strategy for generating new structure-activity data on relaxins.

INSL5 (insulin-like peptide 5) is a two-chain peptide hormone related to insulin and relaxin. It was recently discovered through searches of expressed sequence tag databases and, although the fulfil biological significance of INSL5 is still being elucidated, high expression in peripheral tissues such as the colon, as well as in the brain and hypothalamus, suggests roles in gut contractility and neuroendocrine signalling. INSL5 activates the relaxin family peptide receptor 4 with high potency and appears to be the endogenous ligand for this receptor, on the basis of overlapping expression profiles and their apparent co-evolution. In the present Study, we have used solution-state NMR to characterize the three-dimensional structure of synthetic human INSL5. The structure reveals an insulin/relaxin-like fold with three helical segments that are braced by three disulfide bonds and enclose a hydrophobic core. Furthermore, we characterized in detail the hydrogen-bond network and electrostatic interactions between charged groups in INSL5 by NMR-monitored temperature and pH titrations and Undertook a comprehensive structural comparison with other members of the relaxin family, thus identifying the conserved structural features of the relaxin fold. The B-chain helix, which is the primary receptor-binding site of the relaxins, is longer in INSL5 than in its close relative relaxin-3. As this feature results in a different positioning of the receptor-activation domain Arg(B23) and Trp(B24), it may be an important contributor to the difference in biological activity observed for these two peptides. Overall, the structural Studies provide mechanistic insights into the receptor selectivity of this important family of hormones. 

The human relaxin family comprises seven peptide hormones with various biological functions mediated through interactions with G-protein-coupled receptors. Interestingly, among the hitherto characterized receptors there is no absolute selectivity toward their primary ligand. The most striking example of this is the relaxin family ancestor, relaxin-3, which is an agonist for three of the four currently known relaxin receptors: GPCR135, GPCR142, and LGR7. Relaxin-3 and its endogenous receptor GPCR135 are both expressed predominantly in the brain and have been linked to regulation of stress and feeding. However, to fully understand the role of relaxin-3 in neurological signaling, the development of selective GPCR135 agonists and antagonists for in vivo studies is crucial. Recent reports have demonstrated that such selective ligands can be achieved by making chimeric peptides comprising the relaxin-3 B-chain combined with the INSL5 A-chain. To obtain structural insights into the consequences of combining A-and B-chains from different relaxins we have determined the NMR solution structure of a human relaxin-3/INSL5 chimeric peptide. The structure reveals that the INSL5 A-chain adopts a conformation similar to the relaxin-3 A-chain, and thus has the ability to structurally support a native-like conformation of the relaxin-3 B-chain. These findings suggest that the decrease in activity at the LGR7 receptor seen for this peptide is a result of the removal of a secondary LGR7 binding site present in the relaxin-3 A-chain, rather than conformational changes in the primary B-chain receptor binding site. 

Warfarin is one of the most commonly used drugs in anticoagulent therapy. Notwithstanding its wide use, achieving correct dosage is often a major challenge due to its narrow therapeutic window.[1] The bioavailability of warfarin is believed to be greatly influenced by the environment-dependent composition of the ensemble of isomers present. While the different structures of warfarin have been discussed in earlier publications,[2] details of the mechanism underlying the formation of the cyclic hemiacetal (Figure 1) had not yet been investigated.

Figure 1. Cyclization reaction of warfarin.

Figure 2. Transition state in presence of one water molecule.

We have now studied the reaction by means of density functional calculations. Comparison of results from calculations performed on the isolated warfarin molecule and in presence of water molecules (compare Figure 2) highlight the importance of intermolecular interactions in the key proton transfer step for the reaction to proceed. A viable model for the mechanism underlying the isomerisation shall be presented.

References

[1]             J. Ansell, J. Hirsh, L. Poller, H. Bussey, A. Jacobsen and E. Hylek, Chest, 126, 204S (2004).

[2]            B. C. G. Karlsson, A. M. Rosengren, P. O. Andersson and I. A. Nicholls, J. Phys. Chem. B, 111,10520 (2007).

The anticoagulent drug warfarin exhibits chameleon-like isomerism, where the environment-dependent composition of the ensemble of structures greatly influences its bioavailability. Here, the mechanism of conversion between the major isomeric forms is studied. The dramatic differences in transition state energies, as determined by density functional calculations, highlight the necessity for the involvement of intermolecular interactions in the key proton transfer step. A viable model for the mechanism underlying the isomerization reactions is presented.

Density functional calculations are employed in the study of the C-C bond formation step of an aldol reaction in presence of a series of metals. Focus was placed on first row d-block metals that have been used in catalysis of direct aldol reactions. The obtained energy profiles are analysed in order to differentiate between factors governing catalysis. Results demonstrate a major influence of d-orbital occupation, and suggest some of the so far less commonly used metals as promising candidates for development of new catalytic systems.